Skill: genome-alignment-qc

Use When

• User wants to evaluate BAM quality before variant calling.
• User needs to assess coverage depth and uniformity across the genome or target regions.
• User wants to check mapping rate, duplication rate, and insert size distribution.
• User needs to calculate on-target rate for whole-exome sequencing (WES).
• User wants to compare QC metrics across samples to detect batch effects.

Inputs

• Required:
  • BAM file with index (.bam + .bai).
• Optional:
  • Target BED file (for WES on-target metrics).
  • Reference FASTA (required for picard metrics).
  • Output directory (default: current directory).
  • Per-base coverage flag (enables mosdepth per-base output; default: off).

Workflow

1. Run samtools stats and samtools flagstat for mapping summary.
2. Run samtools idxstats for per-chromosome read counts.
3. Run mosdepth for coverage distribution (genome-wide, or restricted to target BED).
4. If target BED provided: compute on-target rate and per-target coverage statistics.
5. Run picard CollectInsertSizeMetrics for insert size distribution.
6. Run picard CollectAlignmentSummaryMetrics for detailed alignment statistics.
7. Compile summary: mapping rate, duplicate rate, mean coverage, coverage uniformity (CV), median insert size, on-target rate (if WES).
8. Flag metrics outside recommended thresholds (see knowledge/sources/genomics/quality-thresholds.md).

Output Contract

• samtools stats and samtools flagstat output files.
• mosdepth coverage files (.mosdepth.global.dist.txt, .per-base.bed.gz if enabled).
• Picard insert size metrics file and histogram.
• Picard alignment summary metrics file.
• Compiled QC summary with key metrics and threshold flags.

Limits

• samtools, mosdepth, and picard must be installed and available on $PATH.
• mosdepth per-base output can be very large for WGS (~10 GB for 30x human).
• Picard requires a Java runtime (Java 8+).
• Reference FASTA is required for picard metrics but not for samtools/mosdepth.
• This skill reports metrics for a single BAM; multi-sample comparison is out of scope.
• Common failure cases:
  • BAM index (.bai) missing or out of date relative to the BAM file.
  • Target BED file using different chromosome naming convention than the BAM (e.g., chr1 vs 1).
  • Picard failing due to missing reference FASTA sequence dictionary (.dict).